PROJECT SUMMARY/ABSTRACT: Influenza, ebola, dengue and zika are only a few of the viruses which caused past or ongoing pandemics and threaten recurrence. All of these viruses package their infectious genomes in a cell-derived membrane envelope decorated with viral proteins that mediate infectious cell entry. Due to pressures for mechanistic economy imposed by their small size, viruses often combine in a single protein different interdependent functions. Yet they retain extreme adaptability ? they evolve rapidly under changing conditions. Viral cell-entry proteins combine distinct protein domains with membrane fusion and target cell attachment functions. Membrane fusion delivers viral genomes to the cell interior by merging the viral envelope with a cell membrane. The attachment domains bring viruses to the fusion target. They also serve to stabilize fusion domains in the extracellular environment, which retards fusion within endosomes (sites of fusion inside cells) ? they act as fusion repressors. Thus productive cell entry requires a delicate balance of protein's stability and functional plasticity. One source of changing external pressures on cell entry are neutralizing antibodies produced by the immune system. Another source are mechanistic pressures of zoonotic adaptations, when entry proteins adjust preference from animal to human receptors, and alter fusion protein stability as required for human-to-human transmission. How is the extreme adaptability achieved by a system that is constrained both by the changing environment and mechanistic economy? In my past work, I achieved unprecedented, molecular-level resolution of influenza membrane fusion by combining 1) single-virion real-time imaging of membrane fusion of authentic virions with planar membrane bilayers, 2) computational and theoretical modeling of time-delay distributions to various fusion intermediates, and 3) structure-based mutagenesis. This work offered the first glimpse into how adaptability of membrane fusion might be achieved through combined action among multiple fusion proteins on a virus. Hundreds of fusion proteins on a single influenza virus particle contact the target membrane, but only several neighboring ones mediate fusion. I have identified independent avenues available to influenza to fine-tune fusion, two of which affect either the available pool of active fusion proteins on the virion surface or the required number of fusion-protein neighbors. In this proposal, I seek to 1) determine the molecular mechanisms allowing adaptation of the interdependent cell-entry functions of influenza, and 2) define the constraints limiting evolvability of the influenza virus cell-entry functions. I will build upon my combined approaches to enable a holistic molecular picture of membrane fusion and membrane attachment/fusion-repression functions of intact HAs on authentic virions and in near native conditions of purified endosomes. By defining evolutionary rules, entry-protein functional changes, and their co-dependence, we might improve our predictive ability for viral strains with high pandemic potential. Fundamental principles deduced for influenza are likely to extend to other viral cell-entry systems.